Mix up genomic library with cell-free expression system and oil.
End up with oil droplets with DNA plus expression system.
Advantages? Not sure
Apparently better than other in vitro selection techniques such as phage display/ribosome display/mRNA peptide fusion/SELEX (would like to read about these too)
----
Paper on DNA shuffling of Fe-Fe hydrogenases
-> heterologous expression of h2ase in E coli:D
Looks to be similarly possible for Ni-Fe.
---
Directed evolution of oxygenases:
Table 1 compares selection methods
FACS pretty good (10^7). Phage display probs wouldn't work cos the protein wouldn't fold properly. Solid media a bit iffy, but I think it would work well if there was a selection pressure on the cells.
----
Monday, February 22, 2010
Tuesday, February 16, 2010
Enzyme (re)design: lessons from natural evolution and
computation
John A Gerlt1 and Patricia C Babbitt
"Aggressive enzyme engineering". Instead of making small changes, tear off massive chunks of the protein and add on new loops.
Use computers (RosettaMatch) to work out what loops will work well on what scaffold.
Follow it with directed evolution to optimise function.
computation
John A Gerlt1 and Patricia C Babbitt
"Aggressive enzyme engineering". Instead of making small changes, tear off massive chunks of the protein and add on new loops.
Use computers (RosettaMatch) to work out what loops will work well on what scaffold.
Follow it with directed evolution to optimise function.
Wednesday, February 10, 2010
"Enzyme (re)design: lessons from natural evolution and
computation"
- functionally diverse superfamilies: same catalytic method, different reactions
- can create new catalysts by taking an original enzyme and modifying its specificity (same catalytic method)
- usually occurs at expense of original functionality
- other option: some enzymes are promiscuous: use directed evolution to favour one reaction.
computation"
- functionally diverse superfamilies: same catalytic method, different reactions
- can create new catalysts by taking an original enzyme and modifying its specificity (same catalytic method)
- usually occurs at expense of original functionality
- other option: some enzymes are promiscuous: use directed evolution to favour one reaction.
Thursday, January 21, 2010
Wednesday, January 20, 2010
9000 turnovers per second = from Desulfovibrio vulgaris.
Glucose transporter: "
http://www.kazusa.or.jp/codon/ - "Count Codon" service
Inosine = universal base pair
Stratagene Mutazyme = good system for ePCR (less mutational bias - inherently error prone polymerase)
Functional proteins from a random-sequence library: completely random sequence of DNA, combined with mRNA display (?) allowed the generation of a novel ATP binding protein:O
Directed Evolution of Novel Protein Functions:
- From a theoretical perspective, the frequency of functional proteins in the entire protein sequence space is high enough that novel proteins could be generated simply by random methods (but still, low frequency). Pretty cool! If you can come up with a screening mechanism, then you can generate proteins that can do anything:O
- Recognising that it may take several sequential mutations to achieve a desired function, Chen and Zhao developed a screening methodology that involves iterative experiments that attempt to develop a function protein through a series of logical, intermediate steps
- To develop novel proteins, only a small number of mutations are necessary and these are normally localised to the active site
"GC clamp" - add a GC rich region next to sequence of interest. Use in primers to enhance binding to sequence (GC bonds = stronger)
Directed evolution of biocatalytic processes:
- Improvements in one trait may be offset by decline in another trait
- In order to generate useful industrial biocatalysts, screening conditions in directed evolution experiments should closely mimic those that will be found in the industrial setting. Can perform microscale experiments that can be scaled up to large bioreactors
- It's quite hard to ligate mutagenised DNA fragments into a plasmid to transform an E coli - ie you lost a fair bit of your mutation realm simply due to the inefficiency of ligation and transformation
Directed evolution of industrial enzymes
- 'you get what you screen for'
Glucose transporter: "
Trophic Conversion of an Obligate Photoautotrophic Organism Through Metabolic Engineering"
http://www.kazusa.or.jp/codon/ - "Count Codon" service
Inosine = universal base pair
Stratagene Mutazyme = good system for ePCR (less mutational bias - inherently error prone polymerase)
Functional proteins from a random-sequence library: completely random sequence of DNA, combined with mRNA display (?) allowed the generation of a novel ATP binding protein:O
Directed Evolution of Novel Protein Functions:
- From a theoretical perspective, the frequency of functional proteins in the entire protein sequence space is high enough that novel proteins could be generated simply by random methods (but still, low frequency). Pretty cool! If you can come up with a screening mechanism, then you can generate proteins that can do anything:O
- Recognising that it may take several sequential mutations to achieve a desired function, Chen and Zhao developed a screening methodology that involves iterative experiments that attempt to develop a function protein through a series of logical, intermediate steps
- To develop novel proteins, only a small number of mutations are necessary and these are normally localised to the active site
"GC clamp" - add a GC rich region next to sequence of interest. Use in primers to enhance binding to sequence (GC bonds = stronger)
Directed evolution of biocatalytic processes:
- Improvements in one trait may be offset by decline in another trait
- In order to generate useful industrial biocatalysts, screening conditions in directed evolution experiments should closely mimic those that will be found in the industrial setting. Can perform microscale experiments that can be scaled up to large bioreactors
- It's quite hard to ligate mutagenised DNA fragments into a plasmid to transform an E coli - ie you lost a fair bit of your mutation realm simply due to the inefficiency of ligation and transformation
Directed evolution of industrial enzymes
- 'you get what you screen for'
Monday, January 18, 2010
Ultrahigh-Throughput FACS-Based Screening for Directed Enzyme Evolution
Wiley InterScience :: Article Full Text HTML: "Ultrahigh-Throughput FACS-Based Screening for Directed Enzyme Evolution"
- use GFP as reporter gene, then run through FACS machine.
SCOPE:
Shuffle non-homologous proteins
- use GFP as reporter gene, then run through FACS machine.
SCOPE:
Shuffle non-homologous proteins
Saturday, January 9, 2010
Ralstonia eutropha TF93 Is Blocked in Tat-Mediated Protein Export
Use Phenazine methosulfate to visualise H2ase activity?
What's the Sec pathway in E coli?
broad-host-range vector pGE151
Expression of hydrogenase in Alcaligenes spp. is altered by interspecific plasmid exchange.
Doubling time of Ralstonia eutropha = 230 minutes
Co-translocation of a Periplasmic Enzyme Complex by a Hitchhiker Mechanism through the Bacterial Tat Pathway
Tat signal sequence on small subunit -> small + large getting transported
Sec pathway -> unfolded export
The Photobiological Production of Hydrogen: Potential Efficiency and Effectiveness as a Renewable Fuel
max efficiency with hydrogenase + water splitting = 40.7%
Screening technique patent
holy shit..so fucking clever. Couple hydrogenase expression with h2 uptake sensor and link that with expression of reporter gene!
HupR = regulator of uptake hydrogenase. Splice GUS in (not claimed by patent) and stick some X-gal plus IPTG in the media and there you go! Blue fucking cells! Brilliant!
Escherichia coli hydrogenase 3 is a reversible enzyme possessing hydrogen uptake and synthesis activities
E coli h2ase = oxygen sensitive but maybe a good target for directed evolution>?
Development of Selection and Screening Procedures for Rapid Identification of H2-Producing Algal Mutants with Increased O2 Tolerance
Use of a chromagenic film to directly detect H2 production
The bacterial tryptophan reverse mutation assay with Escherichia coli WP2
Another possible screening mechanism? Couple h2ase 1/2 (uptake enzymes) with tryptophan synthesis enzyme lacking in E coli WP2 strain. Cells producing H2 will also produce the trp enzyme and thus be viable.
Protein engineering of hydrogenase 3 to enhance hydrogen production
- ePCR followed by gene shuffling followed by saturation mutagenesis at the change site was very effective in getting enhanced mutants
- They cultured anaerobically. Maybe aerobic culture would promote oxygen insensitive h2ase mutants? Use uptake h2ase from R eutropha to screen?
Use Phenazine methosulfate to visualise H2ase activity?
What's the Sec pathway in E coli?
broad-host-range vector pGE151
Expression of hydrogenase in Alcaligenes spp. is altered by interspecific plasmid exchange.
Doubling time of Ralstonia eutropha = 230 minutes
Co-translocation of a Periplasmic Enzyme Complex by a Hitchhiker Mechanism through the Bacterial Tat Pathway
Tat signal sequence on small subunit -> small + large getting transported
Sec pathway -> unfolded export
The Photobiological Production of Hydrogen: Potential Efficiency and Effectiveness as a Renewable Fuel
max efficiency with hydrogenase + water splitting = 40.7%
Screening technique patent
holy shit..so fucking clever. Couple hydrogenase expression with h2 uptake sensor and link that with expression of reporter gene!
HupR = regulator of uptake hydrogenase. Splice GUS in (not claimed by patent) and stick some X-gal plus IPTG in the media and there you go! Blue fucking cells! Brilliant!
Escherichia coli hydrogenase 3 is a reversible enzyme possessing hydrogen uptake and synthesis activities
E coli h2ase = oxygen sensitive but maybe a good target for directed evolution>?
Development of Selection and Screening Procedures for Rapid Identification of H2-Producing Algal Mutants with Increased O2 Tolerance
Use of a chromagenic film to directly detect H2 production
The bacterial tryptophan reverse mutation assay with Escherichia coli WP2
Another possible screening mechanism? Couple h2ase 1/2 (uptake enzymes) with tryptophan synthesis enzyme lacking in E coli WP2 strain. Cells producing H2 will also produce the trp enzyme and thus be viable.
Protein engineering of hydrogenase 3 to enhance hydrogen production
- ePCR followed by gene shuffling followed by saturation mutagenesis at the change site was very effective in getting enhanced mutants
- They cultured anaerobically. Maybe aerobic culture would promote oxygen insensitive h2ase mutants? Use uptake h2ase from R eutropha to screen?
Thursday, January 7, 2010
"Recombinant and in vitro expression systems for hydrogenases: new frontiers in basic and applied studies for biological and synthetic H2 production"
heterologous expression of FeFe h2ases quite mature but doesn't look as though anyone's tried synthesising the genes with E coli codon optimisation. Might be a 'Plan B'/second approach: try to engineer O2 resistance
Or how about Shewanella oneidensis? No - it's a facultative anaerobe
R eutropha culture: ATCC 17699 Grows at 26deg c $195
heterologous expression of FeFe h2ases quite mature but doesn't look as though anyone's tried synthesising the genes with E coli codon optimisation. Might be a 'Plan B'/second approach: try to engineer O2 resistance
Or how about Shewanella oneidensis? No - it's a facultative anaerobe
R eutropha culture: ATCC 17699 Grows at 26deg c $195
Reading:
"Biological hydrogen production; fundamentals and limiting processes "
- make algae more efficient by reducing number of antennae chlorophyll molecules (majority of photons wasted)
http://jb.asm.org/cgi/content/full/187/18/6590?view=long&pmid=16159796#R18
- really good for info about heterologous expression
Tat paper:
- broad-host-range plasmid pEDY309 works for R eutropha
- spot mating btw E coli and R eutropha
"Biological hydrogen production; fundamentals and limiting processes "
- make algae more efficient by reducing number of antennae chlorophyll molecules (majority of photons wasted)
http://jb.asm.org/cgi/content/full/187/18/6590?view=long&pmid=16159796#R18
- really good for info about heterologous expression
Tat paper:
- broad-host-range plasmid pEDY309 works for R eutropha
- spot mating btw E coli and R eutropha
Wednesday, January 6, 2010
ideas:
- isolate h2ase subunit of hydrogenase holoenzyme (get electrons from direct coupling of PSII/PSI)
how fast is the SH?
- isolate h2ase subunit of hydrogenase holoenzyme (get electrons from direct coupling of PSII/PSI)
how fast is the SH?
NiFe h2ase: 65 μmol/minmg
FeFe h2ase: 5400 μmol/minmg /9000 turnovers per second(90 times faster!)
Biomimics = ~same as NiFe (Nature of hydrogen interactions with Ni(II) complexes containing cyclic phosphine ligands with pendant nitrogen bases)
- membrane bound hydrogenase: 70 turnovers per second (same order of magnitude)Directed evolution of Ralstonia eutropha Soluble Hydrogenase enzyme
Target genes: HoxH and HoxY (hydrogenase genes)
Strategy:
Use site saturation mutagenesis at the active site of the SH, together with broad spectrum PCR error prone mutagenesis (?) to induce mutations in E-coli optimised synthetic DNA sequence. Then transfect Ralstonia eutropha culture with the DNA and screen for recombinant (endogenous genes replaced) hosts which exhibit improved hydrogenase activity (acid agar test).
1. Order materials (culture, primers for mutagenesis, genes)
2. Grow R eutropha and E coli cultures (->what pH does it like?)
3. Plate out in bicycle spoke fashion on acid agar plates with and without universal indicator to evaluate screening technique
4. Perform mutagenesis
Strategy:
Use site saturation mutagenesis at the active site of the SH, together with broad spectrum PCR error prone mutagenesis (?) to induce mutations in E-coli optimised synthetic DNA sequence. Then transfect Ralstonia eutropha culture with the DNA and screen for recombinant (endogenous genes replaced) hosts which exhibit improved hydrogenase activity (acid agar test).
1. Order materials (culture, primers for mutagenesis, genes)
2. Grow R eutropha and E coli cultures (->what pH does it like?)
3. Plate out in bicycle spoke fashion on acid agar plates with and without universal indicator to evaluate screening technique
4. Perform mutagenesis
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